Cytomegalovirus (CMV) is a member of the herpes virus family, causing clinical illness of particular importance in neonates, immunocompromised, and immunosuppressed patients.
Chou and Dennison (J. Infect. Dis. 163:1229-1234, 1991) disclose the nucleotide sequence of part of the envelope glycoprotein B gene of human CMV that encodes epitopes recognized by virus-neutralizing monoclonal antibodies for 12 distinct clinical isolates of CMV.
U.S. Pat. No. 4,731,325, issued 15 Mar. 1988, discloses restriction fragments of CMV DNA in sandwich hybridizations assays.
U.S. Pat. No. 4,762,780, issued 9 Aug. 1988, discloses the use of restriction fragments of CMV DNA which do not cross-hybridize with human DNA for use in hybridization assays for CMV.
EPA 0271201, filed 2 Nov. 1987, discloses the use of cloned restriction fragments of CMV DNA for use in hybridization assays for CMV.
PCT WO 91/02091, filed 9 Aug. 1990, discloses probe sequences to detect and distinguish herpesviruses, including a 31 residue oligonucleotide probe sequence for CMV detection.
Spector et al. (Clin. Chem. 35/8:1581-1587,1989) disclose a .sup.32 P-labeled RNA probe and three single-stranded DNA oligomer capture probes complementary to mRNA encoding the CMV late matrix protein for use in a "target cycling" assay, and four 20-oligonucleotide primers and two 40-oligonucleotide probes for PCR amplification of CMV.
Commonly owned U.S. Pat. No. 4,868,105, issued 19 Sep. 1989, describes a solution phase nucleic acid sandwich hybridization assay in which analyte nucleic acid is first hybridized in solution to a labeling probe set and to a capturing probe set in a first vessel. The probe-analyte complex is then transferred to a second vessel that contains a solid-phase-immobilized probe that is substantially complementary to a segment of the capturing probes. The segments hybridize to the immobilized probe, thus removing the complex from solution. Having the analyte in the form of an immobilized complex facilitates subsequent separation steps in the assay. Ultimately, single stranded segments of the labeling probe set are hybridized to labeled probes, thus permitting the analyte-containing complex to be detected via a signal generated directly or indirectly from the label.
Commonly owned European Patent Application (EPA) 883096976 discloses a variation in the assay described in U.S. Pat. No. 4,868,105, issued 19 Sep. 1989, describes the signal generated by the labeled probes is amplified. The amplification involves the use of nucleic acid multimers. These multimers are branched polynucleotides that are constructed to have a segment that hybridizes specifically to the analyte nucleic acid or to a nucleic acid (branched or linear) that is bound to the analyte and iterations of a second segment that hybridize specifically to the labeled probe. In the assay employing the multimer, the initial steps of hybridizing the analyte to label or amplifier probe sets and capturing probe sets in a first vessel and transferring the complex to another vessel containing immobilized nucleic acid that will hybridize to a segment of the capturing probes are followed. The multimer is then hybridized to the immobilized complex and the labeled probes in turn hybridized to the second segment iterations on the multimer. Since the multimers provide a large number of sites for label probe attachment, the signal is amplified. Amplifier and capture probe sequences are disclosed for Hepatitis B virus, Neisseria gonorrhoeae, penicillin and tetracycline resistance in N. gonorrhoeae, and Chlamydia trachomatis.
Commonly owned U.S. application Ser. No. 558,897, filed 27 Jul. 1990, now abandoned in favor of commonly owned copending U.S. application Ser. No. 07/813,588, filed 23 Dec. 1991, describes the preparation of large comb-type branched polynucleotide multimers for use in the above-described solution phase assay. Pertinent information from U.S. Ser. No. 558,897 is disclosed below following the Examples. The combs provide greater signal enhancement in the assays than the smaller multimers.